The Role And Mechanism Of Autophagy In Skin Soft Tissue Expansion

BackgroundSkin soft tissue expansion is a treatment that placing expander beneath the healthy skin soft tissue,harvesting extra skin induced by mechanical stretch from the expander and then using newly added skin repair the defect.Although skin expansion has the advantages of sufficient donor skin,better appearance and texture of the expanded flap,and few damage to the donor skin,it has also been hampered in clinic due to the long period of treatment,the limited skin expansion efficiency,and the high risk of complications during skin expansion.The mechanism of skin expansion has not been elucidated is the reason that limited skin expansion efficiency and the high complication rate of skin expansion have not been effectively improved.Therefore,it is critical to investigate the molecular mechanisms and biological process of skin expansion in order to improve the skin expansion efficiency and reduce the incidence of complications.Mechanical stretch induces skin regeneration during skin expansion.Mechanical stretch stimulation can cause many changes on cell proliferation,cell migration,macrophage polarization,and stem cell differentiation.A growing number of studies have confirmed that mechanical stretch can cause autophagy induction.Furthermore,autophagy has been related to growth and development,cell proliferation and apoptosis,stem cell stemness and differentiation,angiogenesis,collagen synthesis,and so on.Autophagy activation has been demonstrated to promote wound healing and regeneration of tissues or organs such as nerves,hair,liver,dental pulp,bone and skeletal muscle,muscle,heart,and haematopoietic stem cells.Autophagy modulation is regarded as a promising therapeutic target.Currently,it is unclear what role autophagy plays in skin regeneration during tissue expansion.To clarify the mechanism of skin regeneration during tissue expansion,it will be critical to monitor the level of autophagy during skin expansion.Furtherly we explored the effects of autophagy regulation on skin regeneration during skin expansion provides a theoretical foundation as well as new therapeutic targets for improving skin expansion efficiency and reducing the complications in clinic.ObjectiveTo monitor the changes of autophagy during skin expansion and to investigate the role and mechanism of autophagy in the mechanical stretch induced skin regeneration.Methods1)In the first part,1 m L small animal skin expander was implanted under the scalp of SD(Sprague-Dawley)rats to construct a rat scalp expansion model.The experimental groups consisted of sham expansion group and expansion group.The expansion group was divided into five groups: 6 hours after expansion(Expansion 6h),12 hours after expansion(Expansion 12h),24 hours after expansion(Expansion 24h),48 hours after expansion(Expansion 48h),and 72 hours after expansion(Expansion 72h).Hematoxylin-eosin staining was used to observe the histological changes in the sham and expanded skin;PCNA immunofluorescence staining and TUNEL staining were performed to detect cell proliferation and apoptosis in the sham and expanded skin;transmission electron microscopy was used to observe the ultrastructural changes in the skin during expansion;immunofluorescence staining was used to detect LC3-II in the sham and expanded skin;and the expression of LC3-II,Beclin-1,and p62 protein during the skin expansion were detected by protein immunoblotting to monitor the autophagic changes.2)In the second part,we constructed an SD rat scalp expansion model and then chose the autophagy activator rapamycin(Rapa)and the autophagy inhibitor autophinib for regulating autophagy.The rat were divided into control(DMSO)group,autophagy activation(Rapa)group,and rescue(Rapa + Autophinib)group and autophagy inhibition(Autophinib)group according to the use of autophagy activators or inhibitors.The autophagy modulation skin expansion model was then constructed by applying PLO containing various drugs topically during skin expansion.The expanded skin of each group of rats was stained with HE,and the efficiency of skin expansion was evaluated by measuring the tattooed areas of expanded skin,the thickness of the epidermis and dermis of expanded skin in each group.Immunofluorescence staining of PCNA,TUNEL staining,and Western Blot were used to detect cell proliferation and apoptosis;CK15,CK14,and CK10 immunofluorescence staining were used to detect the number of epidermal and hair follicle stem cells;laser Doppler flow imaging(LDBF)and CD34 immunofluorescence staining were used to detect blood perfusion and vascular density.The collagen volume fraction(CVF)was calculated and expression levels of Col I and Col III were detected using Masson staining and Western Blot;the number of M2 macrophages in the expanded skin of each group using immunofluorescence co-staining of CD68 and CD206;and the b FGF,EGF,CTGF,TGF-β,and VEGF expression were measured in each group.3)In the third section,rat scalp expansion model was constructed and we screened differentially expressed genes(DEGs),differentially expressed proteins(DEPs)by transcriptome and proteome sequencing.Then GO analysis and KEGG analysis were performed.We screen the autophagy-related m RNA for cluster analysis and used q PCR to verify the expression of autophagy-related m RNA.The key gene PIK3 cb and signal pathway AKT-m TOR were verified by Western Blot,and the expression levels of Yap and PIK3 cb and the phosphorylation level of AKT-m TOR pathway in sham and expanded skin at different time points(6,12,24,48,72 h)were detected by Western Blot.Results1)In the first part,the rat scalp expansion model was constructed.Compared with the sham group,the expanded skin showed a thicker epidermis(P < 0.001),thinner dermis(P< 0.001),reduced collagen content in the dermis(P < 0.001),and an increased number of PCNA positive cells(P < 0.001)and TUNEL positive cells(P < 0.001).Transmission electron microscopy showed more Autophagosomes in the epidermis and dermis of the expanded skin,and immunofluorescence staining showed more LC3-II positive spots in the expanded skin compared to the sham group,mainly in the epidermis and hair follicles.LC3-II(P < 0.005)and Beclin-1(P < 0.05)protein expression was upregulated and p62(P <0.001)expression was downregulated in expanded skin.The autophagic flow increased and then decreased over time after the expansion stimulus,reaching a peak at 48 h after expansion.Immunofluorescence staining showed that PINK1,PARK2 and LC3-II were mainly localized in the epidermis and hair follicles and were more expressed in expanded skin than in sham group.2)In the second part,among the drug-treated groups,the largest area of marked expanded skin was observed in the rapamycin group(P < 0.001)and the smallest in the autophinib group(P < 0.01);the thickness of expanded epidermis(P < 0.001)and dermis(P < 0.001)were thickest in the rapamycin group and the expanded epidermis(P < 0.05)and dermis(P < 0.01)in the autophinib group were thinnest.The autophagic flow in the expanded skin was consistent with expected: the highest level of autophagic flow was in the rapamycin group and the lowest level of autophagic flow was in the autophinib group;LC3-II immunofluorescence staining showed the same trend as the autophagic flow in the expanded skin from each group,and the positive spots were mainly localized in the epidermis and hair follicles.PCNA immunofluorescence staining showed an increase in cell proliferation in the expanded skin of activating autophagy group(P < 0.001),while TUNEL staining and Western blot for BAX,Cleaved-Caspase 3 and BCL-2 protein showed a decrease in the activating autophagy group(P < 0.001).Autophagy inhibition group treated with autophinib had the opposite effect: cell proliferation decreased(P <0.01)and apoptosis increased(P <0.001).Immunofluorescence staining of CK15,CK14 and CK10 showed the highest number of epidermal and hair follicle stem cells in the expanded skin of activating autophagy group and the lowest in the inhibiting autophagy group.Laser Doppler flow imaging and CD34 immunofluorescence staining showed the highest blood perfusion(P <0.001)and vascular density(P < 0.001)in autophagy-activated expanded skin,while autophagy-inhibited dilated skin had the lowest blood perfusion(P < 0.001)and vascular density(P < 0.05).Masson staining and Col I and Col III protein Western Blot analysis showed increased collagen synthesis(P < 0.001)and increased CVF(P < 0.001)in the autophagy-activated group and decreased collagen synthesis(P < 0.05)and decreased CVF(P < 0.01)in the autophagy-inhibited group.Activating autophagy increased the number of M2 type macrophages in expanded skin(P < 0.001),while autophagy inhibition decreased it(P < 0.01).The expression of b FGF(P < 0.01),EGF(P < 0.01),CTGF(P < 0.01),TGF-β(P < 0.05)and VEGF(P < 0.01)was higher in the autophagy-activated expanded skin than in the DMSO group;the expression of these growth factors was lower in the autophagyinhibited expanded skin than in the DMSO group.3)In the third part,transcriptome sequencing of skin from sham and expansion group identified 1868 differentially expressed genes,including 894 up-regulated genes and 974down-regulated genes;proteome sequencing identified 230 differentially expressed proteins,including 145 up-regulated proteins and 85 down-regulated proteins.The key molecule mediating autophagy and skin regeneration during skin expansion is PIK3 cb and the key pathway is AKT-m TOR.q PCR and Western Blot verified the up-regulation of PIK3 cb expression in expanded skin,which was consistent with the sequencing results;Western Blot verified the phosphorylation level of AKT-m TOR pathway was down-regulated after expansion.Western Blot of skin from sham group and expanded skin harvested at different time points after last expansion showed that YAP and PIK3 cb were elevated after expansion.YAP reached peak at 6 h after expansion and PIK3 cb reached peak at 48 h after expansion consistent with the trend of autophagic flow during skin expansion.Conclusions1)The level of autophagy is upregulated after skin expansion and reaches the peak at48 h after expansion.Autophagy mainly occurred in the epidermis and hair follicles of the expanded skin.2)Activating autophagy during skin expansion can promote skin regeneration by promoting skin cell proliferation,reducing apoptosis,increasing the number of epidermal and hair follicle stem cells,increasing skin blood supply,promoting skin collagen synthesis,improving the polarization of macrophages to M2 type in the skin,and increasing the secretion of b FGF,EGF,CTGF,TGF-β,and VEGF.Autophagy modulation is a potential therapeutic target to improve the efficiency of skin expansion and reduce complications in clinical practice.3)Upregulation of PIK3 cb and downregulation of the AKT-m TOR pathway may be key molecules and signaling pathways mediating autophagy and skin regeneration during skin expansion.